解淀粉芽孢杆菌YP6中碱性磷酸酯酶AP2的生物信息学分析及酶学性质

Bioinformatics Analysis and Characterization of Alkaline Phosphatase AP2 from Bacillus amyloliquefaciens YP6

对解淀粉芽孢杆菌YP6基因组中编码碱性磷酸酯酶(AP2)的基因进行生物信息学分析、异源表达和酶学性质研究。结果表明,该基因长1752 bp,编码583个氨基酸,通过氨基酸序列分析,预测AP2的分子量为66 kDa,等电点为8.62,是一种结构稳定、亲水性高且有信号肽的碱性蛋白;经保守结构域分析和多序列比对,发现AP2具有PhoD超家族,并且含有与其它PhoD酶相同或相似的金属离子结合位点。酶学性质测定结果显示,AP2的最适温度30℃,最适pH 5.5,属于一种新的PhoD酶;在金属离子变化过程中,Mn^(2+)、Ba^(2+)和Co^(2+)始终对AP2有激活作用,Fe2+和Fe3+始终对AP2有抑制作用;不同浓度的EDTA-2Na、L-组氨酸、L-丝氨酸和L-半胱氨酸均能抑制AP2的活性;AP2对底物DPP和pNPP的kcat/Km值分别为1.1×10^(5) L/(mmol·s)和8.8×10^(2) L/(mmol·s)。

A gene encoding alkaline phosphatase(AP2)in the genome of Bacillus amyloliquefaciens YP6 was studied via bioinformatics analysis to determine heterologous expression and enzymatic properties.The results showed that the gene was 1752 bp in length,encoding 583 amino acids.Amino acid sequence analysis showed that AP2 had a molecular mass of 66 kDa with an isoelectric point of 8.62,and it was identified as an alkaline protein with stable structure,high hydrophobicity and signal peptide sequence.Conserved domain analysis and multiple sequence alignment revealed that AP2 could be considered a part of the PhoD superfamily and contained the same or similar metal ion binding sites as other PhoD enzymes.The optimal temperature of AP2 was 30℃and its optimal pH value was 5.5,which corresponded to a new PhoD enzyme.During the metal ion change,Mn^(2+),Ba^(2+)and Co^(2+)always activated AP2,whereas Fe2+and Fe3+always inhibited it.Additionally,different concentrations of EDTA-2Na,L-histidine,L-serine and L-cysteine all inhibited AP2 activity.Using DPP and pNPP as substrates for AP2,the values of kcat/Km were found to be 1.1×10^(5) L/(mmol·s)and 8.8×10^(2) L/(mmol·s),respectively.